E. Davies Jones

Assessment of the shape and molecular size of TSH-TSH receptor complexes

Photoaffinity labelling and analysis under denaturing conditions (SDS-PAGE) have shown that the porcine TSH receptor contains an A subunit (Mr = 47,000) which forms the binding site for TSH and a B subunit (Mr = 25,000) linked to the A subunit by a disulphide bridge. In order to assess the size and shape of the receptor under non-denaturing conditions we have solubilized photoaffinity-labelled porcine TSH receptors using the small micelle-sized detergent sodium deoxycholate and analysed the preparations by sucrose density gradient centrifugation and gel filtration. Under these conditions, the cross-linked TSH-TSH receptor complex showed an S20,w of 6.4 S and a frictional ratio f/f0 of 1.8. These values were consistent with those which might be expected from an elongated protein complex with a molecular weight of about 100,000 (the value obtained by SDS-PAGE). Analysis of another thyroid membrane protein, human thyroid microsomal antigen (Mr = 110,000 by SDS-PAGE) under the same conditions gave an S20,w of 6.0 S and f/f0 = 1.3, suggesting that this protein has a compact structure. The TSH receptor A subunit cross-linked to TSH (Mr = 70,000 by SDS-PAGE) gave an S20,w of 4.6 S and f/f0 = 1.8 and these values could be compared with those obtained for the A subunit alone (S20,w = 3.6 S; f/f0 = 1.4; Mr by SDS-PAGE = 47,000) and TSH alone (S20,w = 2.6 S; f/f0 = 1.6; Mr = 28,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoelectric focusing of the human TSH receptor A subunit

The water soluble A subunit of the human TSH receptor has been shown to have an isoelectric point of 5. As both TSH and TSH receptor antibodies have isoelectric points in the region of 8–10, charge-charge interactions must be of major importance in the binding of hormone or antibody to the TSH receptor A subunit.